Compositions for solubilizing cells and/or tissue

ABSTRACT

Solubilizing compositions are provided. The compositions comprise at least one zwitterionic surfactant and at least one nonionic surfactant. In one embodiment, the compositions may be useful for solubilizing and remodeling and/or removing tissue on or beneath a patient&#39;s skin, optionally in conjunction with the application of energy to a region of interest on the skin. In one embodiment at least one analyte may be collected and analyzed from the solubilized tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 13/473,261, filed on May 16, 2012, which is acontinuation-in-part application of U.S. patent application Ser. No.13/095,639, filed on Apr. 27, 2011. U.S. patent application Ser. No.13/095,639 is a continuation-in-part application of U.S. patentapplication Ser. No. 12/664,994, which was filed on Jun. 29, 2010 as aU.S. National Stage filing of PCT/US2008/072384, filed on Aug. 6, 2008,which, in turn, claims priority to U.S. Prov. Pat. Appl. Ser. No.60/963,773, filed on Aug. 6, 2007, and now expired. U.S. patentapplication Ser. No. 13/095,639 is also a continuation-in-partapplication of U.S. patent application Ser. No. 13/126,105, which wasfiled on Apr. 26, 2011 as a U.S. National Stage filing ofPCT/US2010/024010, filed on Feb. 12, 2010, which, in turn, claimspriority to U.S. Prov. Pat. Appl. Ser. No. 61/152,585, filed on Feb. 13,2009, and now expired. All of these related applications areincorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under federal grantnumber W81XWH-06-01-00400 awarded by the United States Army. The UnitedStates Government has certain rights in this invention.

BACKGROUND

Skin is the “window” to the body. Skin is unique among the body's organsfor several reasons: (1) skin is the largest organ of the human body;(2) skin is directly exposed to the environment; (3) skin is anexcellent excretory organ; (4) skin is the most visible and accessibleorgan of the body; and (5) skin is a highly active immune organ of thebody.

Skin has another important quality: The molecular profile of skin hasinformation that is valuable for physiological monitoring of, amongother things, small organic molecules, proteins, DNA, RNA, and lipids.Much can be learned from skin's molecular profiling. For example,pathogens (e.g., bacteria) that grow on skin may allow for forensicidentification. Skin's molecular profile may reveal environmentalfactors to which the body has been passively exposed. Theseenvironmental factors may range from the mundane, e.g.,. allergens,toxins, and cosmetic products, to the industrial and/or agricultural,e.g., industrial solvents, fertilizers, and pesticides, to thedangerous, e.g., explosives and other warfare agents.

Skin's molecular profile may also reveal factors to which the body hasbeen actively exposed. More particularly, skin's molecular profile mayreveal what the body has consumed. For example, abused substances (e.g.,illegal drugs or narcotics) and therapeutic drugs (e.g., tramadolfluconazole, barbitals, and anabolic steroids) may be found in skinweeks after consumption.

Skin's molecular profile may also aid diagnosis of conditions anddiseases. For example, skin cholesterol is a proxy of the extent ofarterial blocks. Glycation of skin collagen is an indicator of a historyof diabetes. Skin deposition of β-amyloids may indicate the existenceand extent of Alzheimer's disease. And skin globular proteins (e.g.,IgE) may indicate allergies to specific-allergens.

Several methods exist for sampling biomolecules from skin. For example,one current method is skin biopsy. However, skin biopsy is invasive andanalysis is difficult. Practically speaking, skin biopsy is designed forwell-equipped experts and, thus, its use in a point-of-care setting islimited. Another current method for sampling biomolecules from skin,tape stripping, suffers from these same limitations and is generallyunacceptable because of variability in results. Yet another currentmethod for sampling biomolecules from skin is taking a skin swab. Whiledesirable because of its simplicity, a skin swab is superficial in itsdepth of inspection, and qualitative in its results. Finally, tissue hasbeen subjected to ultrasound in the presence of surfactants such assorbitans (“SPANs”), polyoxyethelene sorbitans combined, with fattyacids (“tweens”), cetyl trimethylammonium bromide (“CTAB”), and theirmixtures. See U.S. Pat. No. 6,589,173 issued to Mitragotri et al.However, SPANs, tweens, and CTAB, individually and collectively, havebeen found to be unsuitable to recover skin constituents. Sorbitans andtweens, which are nonionic surfactants, are mild and non-denaturing incharacter, but are ineffective to solubilize skin tissue. CTAB, acationic surfactant, is effective to solubilize skin tissue, butunsuitably denatures proteins, profoundly changing properties ofbiomolecules in solution, rendering them unusable for functionalpurposes.

Along with providing a cornucopia of information, skin can also be ahost to myriad undesirable cosmetic conditions, such as age spots, skintags, seborrheic keratosis, scar tissues, xanthomas, non-canceroushyperproliferative conditions, surface bumps, and scaly patches; andtherapeutic conditions such as skin tumors, actinic keratosis,leukoplakia, and surface cancers relating to Barrett's esophagus andright-colon pre-cancer plaque. For these conditions, solubilization andremodeling or removal may be the primary concern, with or withoutsubsequent diagnostic processing.

A need exists for compositions for skin sampling, as well as for mucosalmembrane and other tissue sampling, which, when used in conjunction withapplied energy, at least partially solubilize such skin, mucosalmembrane, and other tissue. A further need exists to preserve thefunctionality and structural integrity of analytes, includingbiomolecules obtained from the solubilized skin, mucosal membrane, andother tissue.

SUMMARY

In one embodiment a composition is provided, the composition comprising:

a zwitterionic surfactant, comprising:

wherein n=10, 12, 14,16, or 18; and

a non-ionic surfactant, comprising:

H(CH₂)_(a)O(CH₂CH₂O)_(b)H

wherein a=12 or 16, and wherein b=2, 4, 10, 20, or 23.

In one embodiment, n=12 (corresponding toN-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or “DDPS”), a=16,and b=10 (corresponding to polyoxyethylene (10) cetyl ether or “BrijC10”).

In one embodiment, n=14 (corresponding toN-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or “TPS”), a=12,and b=4 (corresponding to polyoxyethylene (4) lauryl ether or “Brij30”). In another embodiment, n=14 (TPS), a=16, and b=2 (corresponding topolyoxyethylene (2) cetyl ether or “Brij 52”). In another embodiment,n=14 (TPS), a=16, and b=10 (Brij C10).

In one embodiment, n=16 (corresponding toN-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or “HPS”), a=12,and b=4 (Brij 30). In another embodiment, n=16 (HPS), a=16, and b=2(Brij 52). In another embodiment, n=16 (HPS), a=16, and b=10 (Brij C10).

In one embodiment, n=18 (corresponding toN-octadecyl-N,N-dimethyl-3ammonio-1-propanesulfonate or “OPS”), a=12,and b=4 (Brij 30). In another embodiment, n=18 (OPS), a=16, and b=2(Brij 52). In another embodiment, a=18 (OPS), a=16, and b=10 (Brij C10).In another embodiment, n=18 (OPS), a=16, and b=20 (corresponding topolyoxyethylene (20) cetyl ether or “Brij 58”).

In one embodiment, a method Is provided for solubilizing, and remodelingand/or removing tissue on or beneath, a patient's skin, comprisingoptionally applying energy to a region of interest on the skin; andcontacting the region with a tissue solubilizing composition.

In another embodiment, a method for recovering analytes from mucosalmembrane, skin, or other tissue is provided, the method comprising:optionally applying energy to a region of interest on the mucosalmembrane, skin, or other tissue containing at least one analyte;contacting the region with a tissue solubilizing composition, therebysolubilizing at least some of the mucosal membrane, skin, or othertissue containing at least one analyte; and collecting the at least oneanalyte from the solubilized mucosal membrane, skin, and other tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying figures, experimental data are given that, togetherwith the detailed description provided below, describe exampleembodiments of the claimed invention.

FIG. 1 Illustrates in graphical form the total protein (mg/ml) recoveredfrom porcine skin when the porcine skin is contacted with variouscombinations of zwitterionic and nonionic surfactants in the presence ofultrasound.

FIG. 2 illustrates in graphical form the soluble protein (mg/ml)recovered from porcine skin when the porcine skin is contacted withvarious combinations of zwitterionic and nonionic surfactants in thepresence of ultrasound.

FIG. 3 illustrates the dependence on concentration of the proteinextraction efficacy from porcine skin of TPS:Brij C10.

FIG. 4 illustrates in graphical form the preserved activity of theintracellular enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) invarious combinations of zwitterionic and nonionic surfactants.

FIG. 5 illustrates in graphical form the combined dependence of solubleprotein extraction efficacy and GAPDH stability in various combinationsof zwitterionic and nonionic surfactants.

FIG. 6 illustrates the total protein recovery from Human EpidermalKeratinocyte (HEK) cells at t=0 (solid bars), t=4 hours at 4° C.(diagonal Bars), and t=4 hours at RT (cross-hatched Bars), using variousreagents.

FIG. 7 illustrates the GAPDH activity measured in HEK cells solubilizedin various reagents, at t=0 (solid bars), t=4 hours at 4° C. (diagonalbars), and t=4 hours at RT (cross-hatched bars).

FIG. 8 illustrates the GAPDH activity measured b HEK cells solubilizedin various reagents, at room temperature at t=4 hours (solid bars), t=24hours (diagonal bars), 72 hours (cross-hatched bars), and 7 days(horizontal bars).

FIG. 9 illustrates the total protein recovered from homogenized mouseskin using various reagents.

FIG. 10 illustrates the specific GAPDH activity measured in homogenizedmoose skin using various reagents.

DETAILED DESCRIPTION

In one embodiment, a composition is provided, the compositioncomprising:

a zwitterionic surfactant, comprising;

wherein n=10, 12, 14, 16, or 18; and

a non-ionic surfactant, comprising:

H(CH₂)_(a)O(CH₂CH₂O)_(h)H

wherein a=12 or 16, and wherein b=2, 4, 10, 20, or 23.

In one embodiment, the zwitterionic-surfactant comprises DPS and thenonionic surfactant comprises at least one of Brij 30, Brij 35, Brij 52,Brij C10, and Brij 58. In one embodiment, the combination of DPS:Brij 30is excluded.

In another embodiment, the zwitterionic surfactant comprises DDPS andthe nonionic surfactant comprises at least one of Brij 30, Brij 35, Brij52, Brij C10, and Brij 58.

In another embodiment, the zwitterionic surfactant comprises TPS and thenonionic surfactant comprises at least one of Brij 30, Brij 35, Brij 32,Brij C10, and Brij 58.

In another embodiment, the zwitterionic surfactant comprises OPS and thenonionic surfactant comprises at least one of Brij 30, Brij 35, Brij 52,Brij C10, and Brij 58, In one embodiment, the combination of HFS:Brij 35is excluded.

In another embodiment, the zwitterionic surfactant comprises OPS and thenonionic surfactant comprises at least one of Brij 30, Brij 35, Brij 52,Brij C10, and Brij 58.

The zwitterionic surfactant and the nonionic surfactant may be dissolvedin a buffer solution. The buffer solution may comprise, for example, oneor more of phosphate-buffered saline (PBS) (pH=7.2-7.6), tris-bufferedsaline (pH=7.4-8.0), tris-hydrochloride (7.0-9.0), andethylenediaminetetraacetic acid (EDTA) (pH=7.4-9.0). Thus, thesurfactant combination may have a pH of greater than about 7.0 in buffersolution, between about 7.0 and 9.0 in buffer solution, between about7.4 and 9.0 in buffer solution, between about 7.4 and 8.0 in buffersolution, and between about 7.2 and 7.6 in buffer solution.

The zwitterionic surfactant and the nonionic surfactant may be presentin a total concentration of between about 0.01% and about 10% (w/v) mthe buffer solution. For example, the zwitterionic surfactant and thenonionic surfactant may be present in a total concentration of about0.01% to about 5% (w/v) in the holier solution, including totalconcentrations of about 0.1% (w/v) to about 2% (w/v) in the buffersolution, about 1% (w/v) in the buffer solution, and about 0.1% (w/v) toabout 0.5% (w/v) in the buffer solution. In one embodiment, thezwitterionic surfactant and the nonionic surfactant are present in atotal concentration of about 0.5% (w/v) In the buffer solution. Inanother embodiment, the zwitterionic surfactant and the nonionicsurfactant may be present in a ratio of about 3:1 to about 1:3 or about3:2 to about 2:3. In one embodiment, the zwitterionic surfactant and thenonionic surfactant may be present in a ratio of about 1:1.

The zwitterionic surfactant nonionic surfactant composition may

have several applications.

For example, in one embodiment, a method is provided for solubilizingand remodeling and/or removing tissue on or beneath a patient's skin,comprising applying energy to a region of interest on the skin; andcontacting the region with a tissue solubilizing composition comprisingat least one of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52,Brij C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58; (3) TBS and at least one of Brij 30,Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least one ofBrij 30, Brij 35, Brij 52, Brij C10, and Brij 58: and (5) OPS and atleast one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58.

In another embodiment, a method for recovering analytes from mucosalmembrane, skin, or other tissue is provided, the method comprising:applying energy to a region of interest on the mucosal membrane, skin,or other tissue containing at least one analyte; contacting the regionwith a tissue solubilizing composition, the tissue solubilizingcomposition comprising at least one of (1) DPS and at least one of Brij30, Brij 35, Brij 52, Brij C10, and Brij 58; (2):DDPS and at least oneof Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; (3) TPS and atleast one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58: (4) HPSand at. least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;and (5) OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58, thereby solubilizing at least some of the mucosal membrane,skin, or other tissue containing at least one analyte; and collectingthe at least one analyte from the solubilized mucosal membrane, skin,and other tissue.

In some embodiments, “other tissue” may include breast, prostate, eye,vagina, bladder, nail, hair, colon, testicles, intestine, lung, brain,pancreas, liver, heart, bone, or aorta wall.

In some embodiments, an “analyte” may include any biomolecule, drag,small molecule, warfare agent, environmental contaminant, microbe, andthe like that is present in or on the tissue and can be extracted fromthe tissue of interest.

In some embodiments, “biomolecules” may include proteins (e.g., diseasebiomarkers such as cancer biomarkers, antibodies: IgE, IgG, IgA, IgD, orIgM, and the like), peptides, lipids (e.g., cholesterol, ceramides, andfatty acids), nucleic acids (e.g., RNA and DNA), small molecules (e.g.,glucose, urea, and creatine), small molecule drugs or metabolites ofsmall molecule drugs, microbes, inorganic molecules, elements, and ions(e.g., iron, Ca²⁺, K⁺, and the like). In some embodiments, thebiomolecule is exclusive of glucose and cancer markers.

In some embodiments, “drugs” may include abused drugs, such as, forexample, cocaine, heroin, methyl amphetamine, and prescription drugstaken in excess of dosage, or taken without a prescription (e.g.,painkillers such as opioids); and therapeutic drugs, such as, forexample, tramadol, fluconazole, barbitals, and anabolic steroids.

In some embodiments, “warfare agents” may include any molecule,compound, or composition of either biological or chemical origin thatmay be used as a weapon. Non-limiting examples of warfare agents includeexplosives, nerve gases (e.g., VX and Sarin), phosgene, toxins, spores(e.g., anthrax), and the like.

In some embodiments, “environmental contaminants” may include anymolecule, compound, or composition that can be detrimental to anindividual, e.g., when at concentrations elevated above a riskthreshold. Examples include water pollutants (e.g., fertilisers,pesticides, fungicides, insecticides, herbicides, heavy metals, andhalides), soil pollutants (e.g., fertilizers, pesticides, fungicides,insecticides, herbicides, heavy metals, and halides), and air pollutants(e.g., NO_(x), SO_(x), greenhouse gases, persistent organic pollutants,particulate matter, and smog).

In some embodiments, solubilizing the target cells and tissue includesthe application of energy. In some embodiments, the energy may beapplied by any number of suitable methods, including mechanical (e.g.,abrasion, shear, vacuum, pressure, suction, ultrasound), optical (e.g.,laser), thermal, and electrical energy. However, in one embodiment, theenergy does not include externally supplied thermal energy (i.e., heat).Suitable energy applicators are disclosed in U.S. patent applicationSer. Nos. 12/664,994, 13/126,105, and 13/095,771, each of which isincorporated by reference herein in its entirely.

The compositions may be used for solubilizing cells for in vitro proteinrecovery. In addition to effective dissolution of cells, thecompositions may provide a benefit of preservation of bioactivity. Thecompositions may also possess the ability to quickly solubilize varioustissues, including those with durable mechanical properties, such asskin. To aid in the preservation of bioactivity, a protease inhibitormay be included in the compositions. However, with or without theaddition of protease inhibitors, the compositions may be able topreserve the biological activity of proteins. The compositions are alsoapplicable in vivo. In particular, the compositions may he able torecover labile phosphoproteins with RPPA, thus opening the possibilityof quickly and non-invasively probing multiple signaling pathways.

In one embodiment, the compositions may be useful as antibacterialcompositions. Thus, a method for inhibiting the growth and. reproductionof bacteria and/or treating a bacterial infection is provided, themethod comprising applying an antibacterial composition to an area thatis subject to attack by the bacteria, the antibacterial compositioncomprising at least one of (1) DPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58; (2) DDPS and at least one of Brij 30,Brij 35, Brij 52, Brij C10, and Brij 58; (3) TPS and at least one ofBrij 30, Brij 35, Brij 52, C10, and Brij 58; (4) HPS and at least one ofBrij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5) OPS and atleast one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58. Themethod may further include the application of energy.

In another embodiment, a method is provided for solubilizing cellsand/or tissues, the method comprising contacting the cells and/ortissues with a composition, the composition comprising at least one of:(1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (2) DDPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52,Brij C10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij52, Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij35, Brij 52, Brij C10, and Brij 58. In one embodiment, proteins,including cytosolic proteins, nuclear proteins, and surface proteins maybe recovered from the solubilized cells and/or tissues. In someembodiments, such as, for example, embodiments where it is desirable topreserve biological activity of the proteins, the composition mayfurther optionally comprise a protease inhibitor.

In one embodiment, the compositions may be used to probe proteinfunctional states and related skin cell signaling pathways. Skin cellsignaling pathways may be stress-induced, and may change over minutes tohours. Phosphorylation is a highly labile post-translationalmodification that regulates many aspects of protein function. Theability to probe these functional states in the epidermis necessitates afast and efficient method to solubilize and isolate phosphoproteins.Thus, in another embodiment, a method is provided for recoveringsignaling proteins from skin cells, the method comprising: contactingthe skin cells with a composition comprising at least one of (1) DPS andat least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; (2)DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52, Brij C10,and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35, Brij 52,Brij C10, and Brij 58, to provide solubilized signaling proteins; andsubjecting the stabilized signaling proteins to reverse phase proteinarray.

In one embodiment, the compositions comprising at least one of: (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful for, among other things,disaggregating, solubilizing, and stabilizing cell components to be usedas disease biomarkers, forensic biomarkers, or both. In one embodiment,the compositions are useful to disaggregate, solubilize, and stabilizecomponents from living tissues in situ from freshly resected tissues,frozen resected tissues, preserved paraffin embedded tissues, tissue andcell extracts and cultured cells derived from cell lines or resectedtissues, and from exogenous agents such as viruses, bacteria, andprions.

In one embodiment, the compositions comprising at least one of; (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful to solubilize, remodel,and remove diseased tissue on or beneath the surface of the skin, orelsewhere within the body. For example, the compositions may be usefulto solubilize, remodel, and remove tissue hosting precancerousconditions such as actinic keratosis, leukoplakia, Barretts esophagus,and right-colon precancer plaque, and surface cancers arising from anyof these precancerous conditions. Other therapeutic uses may includesolubilizing and removing tumors from a variety of surface or deepsites, or treating tumor surgical margins to remove any residual tumorcells at these sites. In some Instances, after treating tumors with thecompositions and solubilizing the constituent tumor markers, the immune,system may detect the dissolved tumor markers and initiate a potentanti-tumor immune response against these markers, leading to regressionof the local tumor, as well as destruction of any systemic tumor cellscarrying the detected tumor markers.

In one embodiment, the compositions comprising at least one of; (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful for treating skin lesionsand damaged skin with therapeutic molecules and drugs that are unable topenetrate an intact outer skin barrier. More particularly, thecompositions described herein may be useful to enhance absorption oftopical therapeutics by removing diseased, tissue, inflammatory cells,and thickened, hyper-keratinized skin that may block access to otherwiseeffective topical therapies. An example of such a use is as apre-treatment for psoriasis topical therapies, since psoriasis lesionstypically have a hardened top layer or hyperkeratosis that inhibitsabsorption. In one embodiment, such treatment may include perturbationof the outer skin barrier using the compositions, and in somecircumstances applied energy, to disrupt the barrier by disaggregationand solubilization of barrier cells and tissues, followed by applicationof the therapeutic molecules and drags directly to the surface of thebarrier-perturbed skin. Example therapeutic molecules and drugs mayinclude, for example, DNA-based, drugs, RNA-based drugs, protein-baseddrugs, peptide-based drugs, lipid-based drugs, carbohydrate-based drugs,small molecule drugs, nanoparticle based drugs, liposome-encapsulateddrugs, and combinations of such classes of drugs.

In one embodiment, the compositions comprising at least one of: (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful for introducing,therapeutic or diagnostic molecules and drugs into the body andbloodstream by disrupting the outer skin layer. In one embodiment, theintroducing may include perturbation of the outer skin barrier using thecompositions, and in some circumstances applied energy, to disrupt thebarrier by disaggregation and solubilization, of barrier cells andtissues, followed by application of the therapeutic or diagnosticmolecules directly to the surface of the barrier-perturbed skin.

In another embodiment, the compositions comprising at least one of: (1)DPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij58; (2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10,and Brij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful to remove malignant andbenign growths and obstructions in other organs in the body or withinthe central and peripheral nervous systems, including, the eye, middle,ear, brain, spinal cord, nerve roots, and ganglia. Since thecompositions may dissociate and dissolve diseased tissue directly afterinjection through a thin needle or catheter, the compositions may allowablative surgery in areas that are not accessible to either open surgeryor even to minimally invasive surgical instruments (such as in thevascular system, including arteries and coronary arteries).

In another embodiment, the compositions comprising at least one of; (1)DPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij58; (2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10,and Brij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful to diminish or reduceIntra-abdominal and peritoneal adhesions by dissolving specific bands ofadherent tissue between intra-abdominal tissues and organs.

In one embodiment, the compositions comprising at least one of; (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful, for wound debridement. Inone embodiment, the treatment may include contacting the compositions,and in some circumstances applied energy, to a wound's surroundingtissue to remove unhealthy tissue, including, for example, necroticeschar and fibrinous slough.

In one embodiment, the compositions comprising at least one of; (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful as a bio-glue to enhancepost-operation healing.

In one embodiment, the compositions comprising at least one of; (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful to promote oral and dentalhygiene. For example, in one embodiment, the compositions may he usefulto soften and/or dissolve hard and soft deposits on teeth and dentures.

In one embodiment, the compositions comprising at least one of; (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, Brij 52,Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35,Brij 52, Brij C10, and Brij 58, may be useful to solubilize, remodel,and remove cosmetically relevant structures on or beneath the surface ofthe skin. For example, in one embodiment, the compositions .may beuseful for treating aged, scarred, and UV-damaged skin, and removingand/or remodeling age spots, skm tags, seborrheic keratosis, scartissues, xanthomas, non-cancerous hyperproliterative conditions, surfacebumps, and scaly patches. In one embodiment, the use may includeperturbation of the outer skin barrier using the compositions, and insome circumstances applied energy, to disrupt the barrier bydisaggregation and solubilization of barrier cells and tissues. In oneembodiment, the compositions may be introduced to deeper layers of skinto facilitate disaggregation, solubilization, and removal of structuresassociated with wrinkling, scarring, or both, of the skin surface. Othercosmetic uses include dermal peel or skin bleaching. The compositionsmay be used to remove discoloration of the skin associated with previousinjury, UV-damage, or aging. In some embodiments, the perturbation maybe followed by application of therapeutic drugs or natural, products andother cosmetic compositions that are believed or known to increase thesmoothness, elasticity, and resilience of skin. Such cosmeticcompositions may include, for example, elastin or its peptides (e.g.,V-V-P-Q), collagen or its peptides, resveratrol, idebenone, co-enzymeQ10, acetyl hexapeptide-3, glycosaminoglycans, palmitoyl pentapeptide-4,sodium hyaluronate, and the like, and combinations thereof.

In one embodiment, cell components recovered using the compositions maybe introduced into biochemical assays to detect, quantify, and identifyspecific biomarkers associated with specific diseases. The biochemicalassays may include all molecular diagnostic assays for detecting DNA,RNA, proteins, peptides, lipids, carbohydrates, and small molecules,both endogenous and exogenous. The biochemical assays to be used includePCR, ELISA, chromatography, gel analysis, electrophoresis. WesternBlots, Southern Blots, Northern Blots, and other methods used inclinical laboratories for identification, of molecular biomarkers ofdisease.

In one embodiment, the compositions comprising at least one of: (1) DPSand at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58;(2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, andBrij 58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, BrijC10, and Brij 58; (4) HPS and at least one of Brij 30 Brij 35, 52, BrijC10, and Brij 58; and (5) OPS and at least one of Brij 30, Brij 35, Brij52, Brij C10, and Brij 58, may be useful in the treatment of mucosal andskin lesions. Such use may involve impregnating an abrasive andabsorbent swab (similar to a for-daily-use facial exfoliating sponge)with one or more of the compositions and applying the swab to the regionof interest with medium pressure in a twisting or back-and-forth motiononto target tissue for a predetermined time. The depth may be controlledby the amount of pressure, as well as application time. After the lesionis dissolved and the released biomarkers are absorbed into the pad, theswab and the dissolved tissue may be collected and sent to, e.g., apathology lab, for biomarker and other analyte analysis.

EXAMPLES

Certain embodiments are described below in the form of examples. It isimpossible to depict every potential application of the invention. Thus,while the embodiments are described in considerable detail, it is notthe intention to restrict or in any way limit the scope of the appendedclaims to such detail, or to any particular embodiment.

Example 1 Materials

DPS, DDPS, TPS, HPS, and OPS were procured from Sigma-Aldrich aszwitterionic surfactants. Brij 35, Brij 52, Brij C10, and Brij 58 wereprocured from Sigma-Aldrich as nonionic surfactants. Brij 30 wasprocured, from TCI America. All solutions were prepared by adding 0.25%w/v zwitterionic surfactant and 0.25% w/v nonionic surfactant (0.25% v/vB30), for a total concentration of 0.5% w/v In PBS (pH=7.4). A positivecontrol was prepared by placing 1% v/v Triton. X-100 (TX-1) in PBS.

Example 2 Protein Recovery from Porcine Skin

Protein was recovered from porcine skin as a model tissue. Skin wasprocured in frozen form from Lampire Biological Laboratories Inc., andstored at −70° C. Two hours before use, skin was thawed at roomtemperature (RT) and cut into small pieces (2.5 cm×2.5 cm). Skin pieceswere stripped off from subcutaneous fat and used without visiblescratches or abrasions. Protein recovery was carried out by mounting theskin piece on a Franz diffusion cell (FDC) assembly (tissue exposurearea of 1.77 cm²; Permegear). The receiver chamber of the FDC was filledwith PBS and the donor chamber was filled with 1 ml of surfactantcombination as a sampling buffer. This buffer also acted as the couplingfluid between the ultrasound transducer and the tissue. Protein recoverywas performed at RT with a 600-W probe sonicator (Sonics & Materials)operating at a frequency of 20 kHz. The ultrasound transducer was placedat a distance of 5 mm from the tissue surface and an. ultrasonicintensity of 2.4 W/cm² at 50% duty cycle was applied for 3 min. Thesampling buffer, how containing solubilized tissue constituents, wasaspirated and kept at −70° C. until analysis.

The solubilization ability for each surfactant formulation disclosedherein was quantified by the concentrations of total protein andsolubilized protein (mg/ml). Supernatants were isolated from the samplesusing a centrifuge operating at 10,000×g and 4° C. for 15 min. Thesolnbllized protein amount was measured In the sample supernatant byusing a colotimetdc detection kit (Micro BCA Protein Assay Kit; Pierce).

Table 1 illustrates the total protein (mg/ml) recovered from porcineskin when the porcine skin is contacted with various combinations ofzwitterionic and nonionic surfactants in the presence of ultrasound.FIG. 1 illustrates the same results in graphical form.

TABLE 1 Brij 30 Brij 35 Brij 52 Brij C10 Brij 58 DPS 1.47 ± 0.26 1.67 ±0.20 1.33 ± 0.46 1.44 ± 0.23 1.69 ± 0.33 DDPS 1.68 ± 0.25 1.90 ± 0.431.95 ± 0.44 2.11 ± 0.29 1.84 ± 0.23 TPS 1.95 ± 0.26 1.79 ± 0.05 2.23 ±0.25 2.33 ± 0.36 1.52 ± 0.19 HPS 2.19 ± 0.05 1.68 ± 0.26 2.32 ± 0.291.98 ± 0.31 1.64 ± 0.39 OPS 2.15 ± 0.54 1.71 ± 0.19 2.22 ± 0.23 2.42 ±0.36 1.88 ± 0.32

Table 2 illustrates the soluble protein (mg/ml) recovered from porcineskin when the porcine skin is contacted with those same combinations ofzwitterionic and nonionic surfactants in the presence of ultrasound.FIG. 2 illustrates the results in graphical form.

TABLE 2 Brij 30 Brij 35 Brij 52 Brij C10 Brij 58 DPS 0.56 ± 0.04 0.99 ±0.30 0.65 ± 0.03 0.91 ± 0.13 1.15 ± 0.07 DDPS 1.02 ± 0.12 1.27 ± 0.191.10 ± 0.12 1.44 ± 0.16 1.39 ± 0.22 TPS 1.40 ± 0.26 1.31 ± 0.15 1.63 ±0.16 1.64 ± 0.33 1.16 ± 0.25 HPS 1.34 ± 0.27 1.29 ± 0.26 1.55 ± 0.131.45 ± 0.18 1.15 ± 0.29 OPS 1.27 ± 0.28 1.09 ± 0.14 1.06 ± 0.25 1.34 ±0.20 1.14 ± 0.16

Example 3 Concentration Impendence

TPS:Brij C10 was investigated for concentration effect. The surfactantconcentration was tested at total concentrations of 0.5%, 1%, and 2% ofTPS:Brij C10, at a 1:1 ratio. FIG. 3 illustrates the dependence onconcentration of TPS:Brij C10 on the soluble and total proteinextraction efficacy from porcine skin.

Example 4 Enzyme Protection

GAPDH from rabbit was procured as a model enzyme from Sigma-Aldrich.CAPDH powder was dissolved in ddH₂O (Milipore) to 90 U/ml anddistributed into an E-tube, then stored at −80° C. until use. Enzymeprotection was carried out by incubation of GAPDH in each combinationdisclosed herein, compared to that in PBS as a positive control. Theconcentration of GAPDH was fixed to 0.5 U/ml. GAPDH was incubated at 37°C. for 10 min. The activity of the GAPDH enzyme (U/ml) was assessed byKDalert™ GAPDH Assay Kit (Ambion Inc, TX, USA). FIG. 4 illustrates ingraphical form the preserved activity of GAPDH in each of the variouscombinations of zwitterionic and nonionic surfactants.

GAPDH activities were multiplied by soluble protein recovery. FIG. 5illustrates in graphical form the combined dependence of soluble proteinextraction efficacy and GAPDH stability in each of the variouscombinations of zwitterionic and nonionic surfactants.

Example 5 Cell Solubilization

HEK cells (HEKa-APF, Invitrogen, Calif., USA) were cultured in a Coming®cell culture treated flask with a Vent Cap (75 cm² Rectangular CantedNeck, Corning). The HEK ceils were grown, between passage 3 to 8 in theEpiLife® Medium with 60 μM calcium added by Human Keratinocyte GrowthSupplement. FIG. 6 illustrates the total protein recovery from HEK cellsat t=0 (solid bars), t=4 hours at 4° C. (diagonal bars), and t=4 hoursat RT (cross-hatched bars), using DPS:Brij 30, TPS:Brij C10, HPS;Brij52, OPS:Brij 52, OPS;Brij C10, and OPS:Brij 58, as compared to TX-1.

Example 6 In vitro Assay

Total protein in solubilized cells was analyzed using a Micro BCAProtein Assay Kit (Fisher Scientific, PA, USA). Trypsinized cells werecounted by hemocytometer (Fisher Scientific, PA, USA) and added to96-well plates, After overnight growth, the medium was removed andreplaced by 100 μl of lysis agents (TX-1, DPS;Brij 30, TPS:Brij C10,HPS:Brij 52, OPS:Brfj 52, OPS;Brij C10, and OPS:Brij 58). FIG. 7Illustrates the GAPDH activity measured in HEK cells solubilized inthose same reagents, at t=0 (solid bars), t=4 hours at 4° C. (diagonalfears), and t=4 hours at RT (crossmatched bars).

For further study of stability, the enzymes were incubated tor one dayat RT, three days af RT, and seven days at RT. FIG. 8 illustrates theGAPDH activity measured at RT, at t=4 hours (solid bars), t=24 hours(diagonal bars), t=72 hours (cross-hatched bars), and 7 days (horizontalbars).

Example 7 Tissue Solubilization

Skin tissue was collected from euthanized mice and stored at −80° C.until use. About 10 mg of each tissue was placed in a 2 ml tube. 400 μlof chilled lysis buffer (TX-1, TPS:Brij C10, HPS:Brij 52, and OPS;BrijC10) and protease inhibitor cocktail tablets (Roche Applied Science, IN,USA) were added to the tube (one tablet per 50 ml; one tablet containsAntipain-dihydrocloride 3 mg, Aprotinin 0.5 mg, Bestatin 0.5 nig,Chymostatin irng, E*64 3 mg, BDTA-Naj-10 mg, Leupeptin 0,5 mg, PeiahloeSC 20 mg, Pepstatin 0.5 mg, and Phosphoramidon 3 mg), Tissue washomogenized using a homogenizer (IKA, NC, USA) at 13,000 rpm for 1 min.FIG. 9 Illustrates the total protein-recovered from homogenized mouseskin using TPS:Brij C10, HPS:Brij 52, and OPS:Brij C10, as compared toTX-1. FIG. 10 illustrates the specific GAPDH activity measured In thehomogenized mouse skin.

To the extent that the term “includes” or “including” is used in thespecification or the claims, it is intended to be inclusive in a mannersimilar to the term “comprising” as that term is interpreted whenemployed as a transitional word in a claim. As used in the specificationand the claims, the singular forms “a,” “an,” and “the” Include theplural. Furthermore, to the extent that the term “or” is employed (e,g,,A or B), it is intended to mean “A or B or both.” Finally, where theterm “about” is used in conjunction with a number, it is intended toinclude ±10% of the number. In other words, “about 10” may mean from 9to 11.

As stated above, while the present application has been illustrated, bythe description of embodiments thereof, and while the embodiments havebeen described in considerable detail, it is not the intention of theapplicants to restrict or in any way limit the scope of the appendedclaims to such detail. Additional advantages and modifications willreadily appear to those skilled in the art with the benefit of thedisclosure provided in this application. Therefore, the application, inits broader aspects, is not limited to the specific details,illustrative examples shown, or any apparatus referred to. Departuresmay be made from such details, examples, and apparatuses withoutdeparting from the spirit, or scope of the general Inventive concept.

What is claimed is:
 1. A composition, comprising: at least onezwitterionic surfactant, represented by the following structuralformula:

and at least one non-ionic surfactant, represented by the followingstructural formula:H(CH₂)_(a)O(CH₂CH₂O)_(b)H, wherein: n is 10, 12, 14, 16, or 18; a is 12or 16; and b is 2, 4, 10, 20, or 23, provided that n, a, and b are notrespectively: 10, 12, and 4; 14, 16, and 10; or 16, 12, and
 23. 2. Thecomposition of claim 1, wherein: n is 12, a is 16, and b is 2; n is 12,a is 16, and b is 10; n is 14, a is 12, and b is 4; n is 14, a is 16,and b is 2; n is 16, a is 12, and b is 4; n is 16, a is 16, and b is 2;n is 16, a is 16, and b is 10; n is 18, a is 12, and b is 4; n is 18, ais 16, and b is 2; n is 18, a is 16, and b is 10; or n is 18, a is 16,and b is
 20. 3. The composition of claim 1, wherein: a is 12 and b is 4;a is 16 and b is 2; or a is 16 and b is
 10. 4. The composition of claim1, wherein n is 12, 14, 16, or
 18. 5. The composition of claim 1,wherein n is 14, 16, or
 18. 6. The composition of claim 1, wherein a+b−nis less than
 21. 7. The composition of claim 1, wherein the compositionhas a pH more basic than about 7.0 in a buffer solution.
 8. Thecomposition of claim 1, wherein the composition has a pH of betweenabout 7.0 and about 9.0 in a buffer solution.
 9. The composition ofclaim 1, further comprising a buffer solution that dissolves thezwitterionic surfactant and the non-ionic surfactant, the zwitterionicsurfactant and the non-ionic surfactant comprising a total concentrationof about 0.01% to about 10% w/v in the buffer solution.
 10. Thecomposition of claim 1, further comprising a buffer solution thatdissolves the zwitterionic surfactant and the non-ionic surfactant, thezwitterionic surfactant and the non-ionic surfactant each being presentin an amount of at least about 0.25% w/v with respect to the buffersolution.
 11. A method for solubilizing and remodeling and/or removingtissue on or beneath a patient's skin, the method comprising: applyingenergy to a region of interest on the skin; and contacting the regionwith a tissue solubilizing composition, the tissue solubilizingcomposition comprising: at least one zwitterionic surfactant,represented by the following structural formula:

and at least one non-ionic surfactant, represented by the followingstructural formula:H(CH₂)_(a)O(CH₂CH₂O)_(b)H, wherein: n is 10, 12, 14, 16, or 18; a is 12or 16; and b is 2, 4, 10, 20, or 23, provided that n, a, and b are notrespectively: 10, 12, and 4; or 14, 16, and
 10. 12. The method of claim11, further comprising: removing at least a portion of the solubilizedregion of interest to leave an exposed area on the skin; and applying tothe exposed area at least one of a therapeutic composition and acosmetic composition.
 13. The method of claim 12, wherein the applyingcomprises applying to the exposed area a therapeutic compositioncomprising at least one of: a DNA-based drug, an RNA-based drug, aprotein-based drug, a peptide-based drug, a lipid-based drug, acarbohydrate-based drug, a small molecule drug, a nanoparticle-baseddrug, and a liposome-encapsulated drug.
 14. The method of claim 12,wherein the applying comprises applying to the exposed area a cosmeticcomposition comprising at least one of: elastin, an elastin-basedpeptide, collagen, a collagen-based peptide, resveratrol, idebenone,co-enzyme Q10, acetyl hexapeptide-3, glycosaminoglycans, palmitoylpentapeptide-4, sodium hyaluronate, and combinations thereof.
 15. Themethod of claim 11, wherein: n is 12, a is 16, and b is 2; n is 12, a is16, and b is 10; n is 14, a is 12, and b is 4; n is 14, a is 16, and bis 2; n is 16, a is 12, and b is 4; n is 16, a is 16, and b is 2; n is16, a is 16, and b is 10; n is 18, a is 12, and b is 4; n is 18, a is16, and b is 2; n is 18, a is 16, and b is 10; or n is 18, a is 16, andb is
 20. 16. The method of claim 11, wherein a+b−n is less than
 21. 17.A method for recovering analytes from mucosal membrane, skin, or othertissue, the method comprising: applying energy to a region of intereston the mucosal membrane, skin, or other tissue containing at least oneanalyte; contacting the region with a tissue solubilizing composition,thereby solubilizing at least some of the mucosal membrane, skin, orother tissue containing at least one analyte; and collecting the atleast one analyte from the solubilized mucosal membrane, skin, and othertissue, wherein the tissue solubilizing composition comprises: at leastone zwitterionic surfactant, represented by the following structuralformula:

and at least one non-ionic surfactant, represented by the followingstructural formula:H(CH₂)_(a)O(CH₂CH₂O)_(b)H. wherein: n is 10, 12, 14, 16, or 18; a is 12or 16; and b is 2, 4, 10, 20, or 23, provided that n, a, and b are notrespectively: 10, 12, and 4; or 14, 16, and
 10. 18. The method of claim17, wherein the analyte comprises one or more of: a protein, a cancerbiomarker, an antibody, a peptide, a lipid, a nucleic acid, a smallmolecule, a microbe, a warfare agent, an environmental contaminant, or adrug.
 19. The method of claim 17, wherein: n is 12, a is 16, and b is 2;n is 12, a is 16, and b is 10; n is 14, a is 12, and b is 4; n is 14, ais 16, and b is 2; n is 16, a is 12, and b is 4; n is 16, a is 16, and bis 2; n is 16, a is 16, and b is 10; n is 18, a is 12, and b is 4; n is18, a is 16, and b is 2; n is 18, a is 16, and b is 10; or n is 18, a is16, and b is
 20. 20. The method of claim 17, wherein a+b−n is less than21.